All patients were informed about the additional risks of a wedge liver biopsy during the bariatric procedure. Blood and liver samples were obtained from consented liver transplantation donators used as Nutlin-3 healthy controls. In addition, to further explore the role of MIC A/B, we also investigated a cohort of 10 patients with NAFL (that is, as a benign form of NAFLD with only simple steatosis). Specimens were split, with one piece stored in 4% formalin solution (Roth, Karlsruhe,
Germany) for subsequent histological examination and the other piece stored in RNA-preserving agent (RNAlater; Ambion Applied Biosystems, Darmstadt, Germany) to determine the expression of selected genes. The study protocol conformed to the ethical guidelines of the
1975 Declaration of Helsinki and was approved by the Institutional Review Board of the University Hospital of Essen. All patients provided written informed consent before enrollment. The patients’ baseline characteristics are given in Table 1. Hematoxylin-eosin staining was performed according to standard techniques. Samples were investigated and quantified according to NAFLD activity score (NAS).17 Steatosis (0–3), hepatocellular ballooning (0–2), and lobular inflammation Barasertib ic50 (0–2) were quantified, respectively. NAS of ≥5 or ≥4 with at least one score for ballooning was defined as NASH. Extent of liver fibrosis was assessed using the modified METAVIR criteria.18 Liver Nutlin-3 mouse tissue was homogenized with a blade homogenizer (IKA, Staufen, Germany) according to standard laboratory procedures.
Total RNA was isolated with the RNeasy mini kit (Qiagen, Hilden, Germany) following the protocol using spin technology. Having spectrophotometrically assured the samples’ purities and adjusted their concentrations, 2 μg of each RNA sample was filled up to a total volume of 100 μL with RNAse-free water. Reverse transcription was performed with the Quanti Tect RT kit (Qiagen) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qrt-PCR) of complementary DNA was performed using the iCycler iQ thermal cycler (Bio-Rad, Hercules, CA) with real-time detection system software 3.0a and Genex software (Bio-Rad) in 30 μL reactions containing 15 μL Quanti Tect Sybr Green master mix (Qiagen), 5 μL complementary DNA, 1 μL forward primer, 1 μL reverse primer (at 10 pmol/μL each), and 8 μL aqua dest. Amplification was performed for 15 minutes at 95°C, followed by 40 cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C. Melting curve data were collected from 95°C to 55°C, at −0.5°C steps for 10 seconds each. Relative gene expressions were calculated from the threshold cycles in relation to housekeeping gene, to untreated controls or healthy donors, respectively.